Application of TRIM25-SNRK-based targeted intervention strategy in the treatment of non-small cell lung cancer

Abstract

This invention relates to the application of a targeted intervention strategy based on the TRIM25-SNRK regulatory axis in the treatment of non-small cell lung cancer (NSCLC). The nucleotide sequences of the TRIM25 mRNA and SNRK mRNA are shown in SEQ ID NO. 1-2, respectively. This invention is the first to discover that combined targeting of the TRIM25-SNRK regulatory axis, compared to simply interfering with TRIM25 expression levels or transfecting exogenous SNRK mRNA expression plasmids, exhibits a more significant antitumor effect on the cell proliferation and migration processes of NSCLC. The siTRIM25 and SNRK-OE combination strategy designed in this invention can simultaneously and specifically knock down TRIM25 mRNA levels and upregulate SNRK mRNA levels. Compared to transfecting siTRIM25 or SNRK-OE alone, cell function experiments show that the combination strategy has a more effective antitumor effect. Therefore, the TRIM25-SNRK regulatory axis can be used as an intervention target for the research and development of antitumor drugs for NSCLC.

Description

Technical Field

[0001] This invention relates to the application of a targeted intervention strategy based on the TRIM25-SNRK regulatory axis in the treatment of non-small cell lung cancer, and belongs to the field of biomedical technology. Background Technology

[0002] Lung cancer, a prevalent malignant tumor worldwide, poses a serious threat to human health due to its highly aggressive nature and significant tumor heterogeneity. Pathologically, lung cancer is mainly classified into two types: small cell lung cancer and non-small cell lung cancer (NSCLC). NSCLC accounts for approximately 85% of clinically diagnosed cases and can be further subdivided into squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Current clinical treatment strategies for NSCLC primarily include surgical resection, adjuvant chemoradiotherapy, targeted therapy, and immunotherapy. Notably, in recent years, targeted drugs targeting specific gene mutations such as EGFR, ALK, KRAS, and BRAF have achieved significant efficacy in personalized NSCLC treatment, effectively prolonging patient survival. However, these targeted drugs commonly induce drug resistance in tumor cells during treatment, which is one of the main factors leading to accelerated disease progression to the terminal stage. Therefore, exploring new driver genes or signaling pathways and developing novel intervention drugs based on these pathways is crucial for improving the prognosis of NSCLC patients.

[0003] Small interfering RNA (siRNA) was initially discovered by David Baulcombe's team in plant research. It is a double-stranded RNA molecule composed of 19-25 nucleotide pairs. In 2001, Thomas Tuschl's team first demonstrated the mRNA interference effect mediated by siRNA in mammalian cells. This groundbreaking discovery promoted the widespread application of siRNA technology in biomedical research and drug development. In the RNA interference technology system, ensuring the specificity and effectiveness of mRNA interference of the target gene is both a core technical point and a key indicator for evaluating the quality of siRNA design. Therefore, designing siRNA molecules with efficient knockdown capabilities and high specificity for specific target genes will provide crucial technical support for functional studies of new driver genes in NSCLC and the development of targeted therapy strategies.

[0004] Gene expression plasmids are synthetic circular DNA molecules that, once transfected into host cells, can replicate autonomously and drive the transcription and translation of specific genes, thereby achieving efficient expression of exogenous target genes within the host cells. Plasmid vectors typically include origin of replication, multiple cloning sites, promoters, terminators, antibiotic resistance genes, and reporter genes. By inserting the coding sequence of the target gene into a plasmid vector and applying transfection technology, the expression of the target gene can be upregulated in target cells. This is of great significance for studying the biological function and potential regulatory mechanisms of target genes in cancer cells. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides the application of a targeted intervention strategy based on the TRIM25-SNRK regulatory axis in the treatment of non-small cell lung cancer.

[0006] The technical solution of the present invention is as follows:

[0007] Application of TRIM25-SNRK regulatory axis as a drug target in the preparation of drugs against non-small cell lung cancer.

[0008] According to a preferred embodiment of the present invention, the TRIM25-SNRK regulatory axis includes the TRIM25 gene and the SNRK gene.

[0009] According to a preferred embodiment of the present invention, the nucleotide sequence of the TRIM25 gene mRNA is shown in SEQ ID NO.1, and the nucleotide sequence of the SNRK gene mRNA is shown in SEQ ID NO.2.

[0010] According to a preferred embodiment of the present invention, the anti-non-small cell lung cancer drug uses the TRIM25 gene and the SNRK gene as intervention targets. While interfering with the expression of the TRIM25 gene, an exogenous SNRK gene is introduced, which can efficiently and specifically downregulate the expression level of the TRIM25 gene and upregulate the expression level of the SNRK gene.

[0011] According to a preferred embodiment of the present invention, the anti-non-small cell lung cancer drug is a combination of an inhibitor that specifically interferes with the expression of the TRIM25 gene and an exogenous expression plasmid that specifically upregulates the expression of the SNRK gene.

[0012] Application of siRNA that specifically interferes with TRIM25 gene expression and exogenous SNRK mRNA expression plasmid that specifically upregulates SNRK gene expression in the preparation of drugs against non-small cell lung cancer.

[0013] According to a preferred embodiment of the present invention, the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the TRIM25 gene is shown in SEQ ID NO.3; and the exogenous SNRK mRNA expression plasmid contains the nucleotide sequence shown in SEQ ID NO.4.

[0014] More preferably, the volume-to-mass ratio of the siRNA that specifically interferes with TRIM25 gene expression to the exogenous SNRK mRNA expression plasmid that specifically upregulates SNRK gene expression is 3:2, in μL / μg.

[0015] An anti-non-small cell lung cancer drug, wherein the anti-non-small cell lung cancer drug contains siRNA that specifically interferes with the expression of the TRIM25 gene and an exogenous SNRK mRNA expression plasmid that specifically upregulates the expression of the SNRK gene.

[0016] According to a preferred embodiment of the present invention, the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the TRIM25 gene is shown in SEQ ID NO.3; and the exogenous SNRK mRNA expression plasmid contains the nucleotide sequence shown in SEQ ID NO.4.

[0017] More preferably, the volume-to-mass ratio of the siRNA that specifically interferes with TRIM25 gene expression to the exogenous SNRK mRNA expression plasmid that specifically upregulates SNRK gene expression is 3:2, in μL / μg.

[0018] Beneficial effects:

[0019] This invention is the first to discover that combined targeting of the TRIM25-SNRK regulatory axis exhibits a more significant antitumor effect on the proliferation and migration of non-small cell lung cancer (NSCLC) cells compared to simply interfering with TRIM25 expression levels or transfecting exogenous SNRK mRNA expression plasmids. The siTRIM25 and SNRK-OE combination strategy designed in this invention can simultaneously and specifically knock down TRIM25 mRNA levels and upregulate SNRK mRNA levels. Compared to transfecting siTRIM25 or SNRK-OE alone, cell function experiments demonstrate that the combination strategy has a more effective antitumor effect. Therefore, the TRIM25-SNRK regulatory axis can be used as an intervention target for the research and development of antitumor drugs for NSCLC. Attached Figure Description

[0020] Figure 1A549 cells were selected and transfected with siNC+Vector or siTRIM25+SNRK-OE. Cells were collected and RNA was extracted. The levels of TRIM25 and SNRK mRNA were detected by reverse transcription-real-time quantitative PCR. The statistical significance of the differences between groups was determined by Student's t test.

[0021] Figure 2 The cells were transfected with siNC+Vector, siTRIM25, SNRK-OE, or siTRIM25+SNRK-OE, respectively. The proliferation capacity of A549 cells was detected by CCK-8 assay, and the statistical significance of the differences between groups was verified by two-way ANOVA.

[0022] Figure 3 A549 cells were selected and transfected with siNC+Vector, siTRIM25, SNRK-OE, or siTRIM25+SNRK-OE, respectively. The migration ability of A549 cells was evaluated using the Transwell assay, and the statistical significance of the differences between groups was determined using the one-way ANOVA. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to specific experimental examples, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the reagents and materials involved in the examples are all commercially available products.

[0024] The human non-small cell lung cancer cell line A549 is available from Shanghai Fuheng Biotechnology Co., Ltd.

[0025] siTRIM25 targeting sequence: 5'-ggtggagcagctacaacaa-3';

[0026] Example 1

[0027] Previous research by the inventors revealed abnormal activity of the TRIM25-SNRK regulatory axis in non-small cell lung cancer (NSCLC) tissues, which is closely related to the malignant progression of NSCLC. Based on this, the inventors discovered that combined intervention of TRIM25 and SNRK expression levels can produce a synergistic anti-tumor effect. This combined intervention strategy provides an innovative approach for developing novel anti-tumor drugs for NSCLC and is expected to play an important role in clinical treatment.

[0028] Example 2

[0029] The nucleotide sequences of TRIM25 mRNA and SNRK mRNA were obtained from the NCBI database, as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. Based on these sequences, siRNAs (siTRIM25) that specifically interfere with the expression level of TRIM25 mRNA were designed and synthesized. The target sequence of siTRIM25 is shown in SEQ ID NO.3, and the control siRNA is siNC. An exogenous SNRK mRNA expression plasmid (SNRK-OE) that specifically upregulates SNRK mRNA was also synthesized. This SNRK-OE plasmid contains the nucleotide sequence shown in SEQ ID NO.4, and its control plasmid is an empty vector plasmid. The empty vector plasmid (Vector) and siNC are both commercially available. The SNRK-OE plasmid was constructed using the Miaoling plasmid platform according to the sequence information, and siTRIM25 was synthesized by Qingke Biotechnology according to the sequence information.

[0030] siNC+Vector or siTRIM25+SNRK-OE were transfected into A549 cells at a growth density of 60%. Cells were collected 48 hours after transfection, and total RNA was extracted. The expression levels of TRIM25 mRNA and SNRK mRNA were detected using reverse transcription-real-time quantitative PCR. Specific results are shown below. Figure 1 As shown.

[0031] Depend on Figure 1 It can be seen that, compared with transfecting siNC+Vector, transfecting siTRIM25+SNRK-OE into A549 cells can significantly downregulate the expression level of TRIM25 mRNA and significantly upregulate the expression level of SNRK mRNA.

[0032] The specific implementation process is as follows:

[0033] (1) A549 cells were plated in 6-well plates. When the cell density reached about 60%, siNC+Vector or siTRIM25+SNRK-OE was transfected into A549 cells at a ratio of 3 μL (10 μM) siRNA volume and 2 μg plasmid mass using Lipofectamine RNAiMAX Reagent (Lifetechnologies, 13778-150) and Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, L3000015), respectively.

[0034] (2) 48 hours after transfection, the cells obtained in step (1) were collected, RNA was extracted using the RNA-Quick Purification Kit (Shanghai Yishan, RN001), the concentration of the extracted RNA was detected using NanoDrop2000, and the integrity of the extracted RNA was assessed by agarose gel electrophoresis.

[0035] (3) Using the RNA obtained in step (2) as a template, cDNA products were synthesized by reverse transcription using the LunaScript™ RT SuperMixKit (NEB, E3010) reverse transcription kit. Power The Green Master Mix (Thermo Fisher Scientific, 4367659) kit was used with a QuantStudio™ 5System (Thermo Fisher Scientific) PCR instrument for quantitative real-time PCR, with ACTB mRNA level as an internal control, according to 2... -ΔΔCT The formula calculates the relative expression levels of TRIM25 mRNA and SNRK mRNA.

[0036] Example 3

[0037] A549 cells were selected and transfected with the same dose of siNC+Vector, SNRK-OE, siTRIM25, or siTRIM25+SNRK-OE, respectively. The proliferation capacity of the transfected cells was evaluated using the CCK-8 assay. The experimental results are as follows: Figure 2 As shown.

[0038] Depend on Figure 2 It was found that transfection with siTRIM25, SNRK-OE, or siTRIM25+SNRK-OE significantly weakened A549 cell growth compared to siNC+Vector. However, transfection with siTRIM25+SNRK-OE exhibited a more significant inhibitory effect on cell proliferation compared to siTRIM25 or SNRK-OE alone. This indicates that combined intervention on TRIM25 and SNRK expression levels has a superior inhibitory effect on tumor cell proliferation compared to targeting either gene expression level alone.

[0039] The specific implementation process is as follows:

[0040] (1) A549 cells were transfected with siNC+Vector, SNRK-OE, siTRIM25 or siTRIM25+SNRK-OE respectively. The dose of Vector and SNRK-OE was 2 μg / well, and the dose of siNC and siTRIM25 was 3 μL (concentration 10 μM) / well. The transfection method and reagents were the same as in Example 2. After 48 hours, the cells were collected and seeded into 96-well plates at a density of 2000 cells / well. The cells were divided into 5 groups (Day 0, 1, 2, 3, 4). Each group had 4 types of transfected cells (Vector+siNC, SNRK-OE, siTRIM25 or siTRIM25+SNRK-OE). Each type of cell was seeded in 3 replicates.

[0041] (2) After the cells obtained in step (1) adhere to the wall (about 2-4 hours), add 10 μL of CCK-8 reagent (TargetMol, C0005) to the Day 0 group, place it in a 37℃ incubator for 1 hour, shake it simply, and then place it in a microplate reader to measure the absorbance value at 450nm. This is the absorbance value of the initial seeded cells (Day 0).

[0042] (3) After completing the initial cell absorbance measurement, the absorbance values ​​of cells in Day 1, 2, 3 and 4 were measured every 24 hours using the same method. The absorbance value of Day 0 was used as a reference to calculate the cell viability changes in each group and the cell proliferation trend in each group.

[0043] Example 4

[0044] A549 cells were selected and transfected with the same dose of siNC+Vector, SNRK-OE, siTRIM25, or siTRIM25+SNRK-OE, respectively. The migration ability of transfected cells was assessed using a Transwell assay. Specific results are shown below. Figure 3 As shown.

[0045] Depend on Figure 3 The results show that co-transfection with siTRIM25+SNRK-OE significantly inhibited the migration ability of A549 cells compared to co-transfection with siTRIM25 or SNRK-OE alone. This indicates that simultaneous intervention on the expression levels of TRIM25 and SNRK genes exhibits superior anti-cancer effects compared to targeting the expression of either gene alone.

[0046] The specific implementation process is as follows:

[0047] (1) Select A549 cells and culture them in 6-well plates. When the cell growth density reaches about 60%, transfect them with siNC+Vector, SNRK-OE, siTRIM25 or siTRIM25+SNRK-OE respectively. The specific method is as described in Example 3.

[0048] (2) After 48 hours of transfection, collect and count the transfected cells obtained in step (1), and adjust the cell concentration to 100,000 cells / mL. Add complete culture medium (600 μL / well) to a 24-well plate and place the Transwell chamber (Corning, 3422) in the well plate;

[0049] (3) Add 200 μL of cell suspension to each Transwell cell chamber and continue culturing in a cell culture incubator (37℃, 5% CO2) for 48 hours;

[0050] (4) Collect the Transwell chambers and discard the culture medium. Wash the cells with PBS. Fix the cells with 4% paraformaldehyde at room temperature for 15 minutes. Then stain with crystal violet (0.1%) overnight to remove the cells that have not migrated from the inside of the chambers. Observe the migrated cells under an inverted microscope and take pictures. Take pictures and record the data and perform statistical analysis.

Claims

1. The use of siRNA specifically interfering with the expression of TRIM25 gene and exogenous SNRK mRNA expression plasmid specifically up-regulating the expression of SNRK gene in the preparation of anti-non-small cell lung cancer drugs, characterized in that, the siRNA specifically interfering with the expression of TRIM25 gene and the exogenous SNRK mRNA expression plasmid specifically up-regulating the expression of SNRK gene are used in combination. The targeted nucleotide sequence of the siRNA specifically interfering with the expression of the TRIM25 gene is shown as SEQ ID NO. 3; the exogenous SNRK mRNA expression plasmid contains a nucleotide sequence shown as SEQ ID NO.

4.

2. Use according to claim 1, wherein The volume-to-mass ratio of the siRNA specifically interfering with the expression of the TRIM25 gene to the exogenous SNRK mRNA expression plasmid specifically up-regulating the expression of the SNRK gene is 3:2, unit: μL / μg.

3. An agent against non-small cell lung cancer, characterized by, The anti-non-small cell lung cancer drug contains the siRNA specifically interfering with the expression of the TRIM25 gene and the exogenous SNRK mRNA expression plasmid specifically up-regulating the expression of the SNRK gene; the targeted nucleotide sequence of the siRNA specifically interfering with the expression of the TRIM25 gene is shown as SEQ ID NO. 3; the exogenous SNRK mRNA expression plasmid contains a nucleotide sequence shown as SEQ ID NO.

4.

4. The anti-non small cell lung cancer drug according to claim 3, wherein The volume-to-mass ratio of the siRNA specifically interfering with the expression of the TRIM25 gene to the exogenous SNRK mRNA expression plasmid specifically up-regulating the expression of the SNRK gene in the anti-non-small cell lung cancer drug is 3:2, unit: μL / μg.

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